Isolated strains of Staphylococcus aureus and vaccines manufactured therefrom

ABSTRACT

The invention provides antigenic compositions for the vaccination of an animal against bovine mastitis caused by infection with  Staphylococcus aureus.  The invention also provides methods for stimulating an animal&#39;s immune system to respond to antigens derived from selected strains of  Staphylococcus aureus  by administering the antigenic compositions of the invention to the animal.

This application claims priority to a 371 PCT/IL98/00627, filed Dec. 29,1998.

FIELD OF THE INVENTION

The present invention relates generally to bovine mastitis infectionscaused by Staphylococcus aureus and, more particularly, to vaccinesderived from selected strains of Staphylococcus aureus.

BACKGROUND

Bovine mastitis is the most important infectious disease affecting boththe quality and quantity of milk production. Staphylococcus aureus(i.e., “S. aureus”) is the prime agent causing bovine mastitis, and itis difficult to eliminate. In different countries, the prevalence of S.aureus mastitis ranges from 10% to 40% of all cows. The infected animalsmay serve as reservoirs of infection endangering other dairy cattle inthe herd (Fox, L. K. and Hancock, D. 1989, “Effects of segregation onprevention of intramammary infection by Staphylococcus aureus”, J. DairySci. 72:540-544).

Recent estimates suggest that the annual production losses due to S.aureus are over 15 million dollars in Israel and over 2 billion dollarsin the USA. The prevalence of S. aureus mastitis in dairy cattle raisesseveral concerns. This bacterium can cause severe damage tomilk-synthesizing tissues, drastically reducing milk production andaltering milk composition. For more information on bovine mastitis andits effects, see, for example: (1) Oliver, S. P., Sordillo, L. M, 1988,“Udder health in the periparturient period”, J Dairy Sd. 71:2584-2606;(2) Postle, D. S., Roguinsky, M., Poutrel, B., 1978, “InducedStaphylococcal infections in the bovine mammary gland”, Am J Vet Res.39:29-35; (3) Sordillo, L. M., Nickerson, S. C and Akers, R. M., 1989,“Pathology of mastitis during lactogenesis: Relationships with bovinemammary structure and function”, J. Dairy Sci. 72: 228-240; (4) Watson,D. L., McColl, M. L., Davies, H. I., 1996, “Field trial of aStaphylococcal mastitis vaccine in dairy herds: clinical, subclinicaland microbiological assessments”, Aust. Vet. J. 74:447-450.

Depending on the duration and the severity of the infection, theproductive performance of dairy cattle may be diminished permanently.Therefore, the development of effective methods of controlling S. aureusmastitis will increase profitability to dairy producers by reducingcosts. So far, post-milking teat disinfection and antibiotic therapy arethe only widely accepted methods of mastitis control (National MastitisCouncil, 1987, “Current concepts of bovine mastitis”, Arlington, Va.).

These methods are not cost-effective due to milk loss during and afterantibiotic therapy. Moreover, antibiotic therapies formulated forintramammary use are generally unsuccessful in eliminating existing S.aureus infections or preventing the establishment of chronic diseases(Ziv, G., 1995, “Treatment of Mastitis: An overview of progress duringthe last ten years”, Proc. The 3rd Internal Mastitis Seminar, Tel Aviv,Israel 2-12).

There is also a growing concern over the presence of drug residues inthe food supply as a consequence of these procedures. To date, cullingchronically infected cows is often the only practical means ofeliminating S. aureus from a herd.

Vaccination is a logical approach for controlling infectious diseases infood producing animals. However, the paucity of information regardingrelevant antigens remains a major deterrent to successful immunizationagainst S. aureus mastitis. To our knowledge, the known, commerciallyavailable S. aureus vaccines have shown limited efficacy under fieldconditions. See, for example:

In the USA:

1) Nickerson, S. C., Owens, W. E., Bodie, R. L, 1993, “Effect of aStaphylococcus aureus bacterin on serum antibody, new infection, andmammary histology in non lactating dairy cows”, J. Dairy Sci.,76:1290-1297;

2) Sears, P. M., Norcross. N. L., Kenny, K., Smith, B., Gonzalez, R. N.,Romano, M. N., 1990, “Resistance to Staphylococcus aureus infections instaphylococcal vaccinated heifers”, Proc. Internatl. Symp. BovineMastitis, Indianapolis, Ind., p. 69.

3) Sordillo, L. M., Nickerson, S. C and Akers, R. M., 1989, “Pathologyof mastitis during lactogenesis: Relationships with bovine mammarystructure and function”, J. Dairy Sci., 72: 228-240; and

4) Yoshida K., Ichiman, Y., Narikawa, S., Evans, G. B., 1984,“Staphylococcal capsular for preventing mastitis in two herds inGeorgia”, J. Dairy Sci., 67:620-627.

In Australia:

1) Watson, D. L., 1984, “Evaluation of attenuated, live staphylococcalmastitis vaccine in lactating heifers”, J. Dairy Sci., 67:2608-2613;

2) Watson, D. L., Schwartzkoff, C. L., 1990, “A field trial to test theefficacy of a staphylococcal mastitis vaccine in commercial dairies inAustralia”, International Symposium on Bovine Mastitis, NationalMastitis Council, Arlington, 73-76;

3) Watson, D. L., 1992, “Vaccination against experimental staphylococcalmastitis in dairy heifers”, Res. Vet. Sci., 53:346-353; and

4) Watson, D. L., McColl, M. L., Davies, H. I., 1996, “Field trial of aStaphylococcal mastitis vaccine in dairy herds: clinical, subclinicaland microbiological assessments”, Aust. Vet. J., 74:447-450.

In Norway:

1) Nordhaug, M. L., Nesse, L. L., Norcross, N. L., Gudding, R., 1994, “Afield trial with an experimental vaccine against Staphylococcus aureusmastitis in cattle. I. Clinical parameters”, J Dairy Sci., 77:1267-1275;

2) Pankey, J. W., et al., 1985, “Evaluation of protein A and acommercial bacterin as vaccines against Staphylococcus aureus mastitisby experimental challenge”, J. Dairy Sci., 68:726-731; and

3) Yoshida K., Ichiman, Y., Narikawa, S., Evans, G. B., 1984,“Staphylococcal capsular for preventing mastitis in two herds inGeorgia”, J. Dairy Sci., 67:620-627.

For the most part, these conventional vaccines have not prevented thedisease and show only a marginal benefit in ameliorating the severityand duration of clinical symptoms of S. aureus mastitis. Traditional S.aureus mastitis vaccines have included killed or attenuated bacteria,toxoids, and cell wall extracts from selected laboratory or fieldstrains. See: (1) Nickerson, S. C.; (2) Sears, P. M.; (3) Watson, D. L.1984; and (4) Watson, D. L., 1992; as cited above.

These previous attempts have not considered the significant variationamong the different strains of S. aureus which cause mastitis.

Attempts to solve this problem are described in U.S. Pat. No. 4,840,794.However this solution has not been satisfactory.

It is, therefore, desirable to develop a vaccine which would overcomethe above disadvantages and would prevent the occurrence of bovinemastitis infection or at least control such infections to a largeextent.

BRIEF DESCRIPTION OF THE DRAWINGS

Additional aspects of the present invention will become evident uponreviewing the non-limiting embodiments described in the specificationand the claims taken in conjunction with the accompanying figures,wherein:

FIG. 1 is an immunoblot of mice sera against BS449 antigen before andafter the administration of an S. aureus vaccine in accordance with theinvention;

FIG. 2 is a time table of two studies involving an S. aureus vaccine inaccordance with the invention;

FIG. 3 is a graphical depiction of results of the studies that are thesubject of FIG. 2; and

FIG. 4 is another graphical depiction of results of the studies that arethe subject of FIG. 2.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

It has been shown that a vaccine which would overcome the abovedisadvantages and would prevent the occurrence of bovine mastitisinfection or is at least control such infections to a large extentcannot be prepared from known strains of S. aureus, and, therefore, newstrains had to be isolated.

In one aspect, the present invention provides the following three fieldstrains of S. aureus, either separately or in combination: strain BS449(i.e., BS); strain ZO3984 (i.e., ZO); and strain VLVL8407 (i.e., VL).For convenience, these strains are collectively referred to herein as“the S. aureus strains”. Each of the three strains may be characterizedby the following basic features:

Strain BS449: β-hemolytic; coagulase positive; and phage type 81;

Strain ZO3984: β-hemolytic; coagulase positive; and phage type 3/A, 3/C,55, and 71; and

Strain VLVL8407: non-hemolytic; coagulase positive; and phage untypable.

Further biochemical and enzymatic features are shown in Table I.Antibiograms are shown in Table II.

TABLE I Biochemical and Enzymatic Characterization VLVL8407 ZO3984 B5449Phage type − 3/A, 3/C, 81 55, 71 Hemolysis − β β Coagulase + + + GLUGlucose + + + FRU Fructose + + + MAL Maltose + + + LAC Lactose + + + TRETrehalose + + + MAN Manitol + + + RAF Rafinose + − − RIB Ribose − − −CEL Celobiose − − − NIT Nitrates (reduction) + + + VP (ActoinProduction) + + + βGal βGalctosidase − − − ArgA Arginine Arylamidase − −− PAL Alkaline Phosphatase + + + PyrA Pyrrolidonyl Arylamidase + + +NOVO Novobiocin (Resistance) − + − Fermentation: SAC Sucrose + + + NAGN-Acetyl-glucosamine + + + TUR Turanose + + + ARA Arbinose − − − βGURβGlucuronidase − − − URE Urease + − + ADH Arginin dihydrolase + + + ODCOrnithine decaboxilase − − − ESC Esculine (hydrolysis) − − −

TABLE II Antibiograms ZO3984 VLVL8407 B5449 METHICILLIN (N)* − − −PENICILLIN (P) − − − OXYTETRACYCLINE (T) ± − − ERYTHROMYCIN (E) ± + +CEPHALOTHIN (CR) + + + NOVOBIOCIN (NB) + + ± NORFLOXACIN (NEF) + + + SXT(SXT) − − − *METHICILLIN represents CLOXACILLIN and NAFCILLIN

The S. aureus strains were deposited under the terms of the BudapestTreaty on Dec. 16, 1997 with the Collection Nationale de Cultures deMicroorganismes (CNCM), Institut Pasteur, 25 Rue du Docteur Roux,F-75724 Paris CEDEX 15. The deposited strains were assigned thefollowing registration numbers:

BS 449 (449) I - 1950 ZO 3984 (84) I - 1951 VLVL 8407 I - 1952

The deposit represents a biologically pure culture of each depositedstrain. Access to the cultures will be available during pendency of thepatent application to one determined by the Commissioner to be entitledthereto under 37 CFR 1.14 and 35 USC 122. All restrictions on theavailability of the cultures to the public will be irrevocably removedupon the granting of a patent based upon the application. However, itshould be understood that the availability of the cultures on depositdoes not constitute a license to practice the subject invention inderogation of the patent rights granted.

The S. aureus strains were chosen from among four-hundred S. aureusfield isolates which were obtained from cows that were known to bechronically infected with bovine mastitis. Antigens from the threeselected S. aureus strains showed a high variability in theirelectrophoretic profiles (e.g., in the range of about 21-36 kilodaltons(kD)). That is, the high electrophoretic variability demonstrated that acombination of antigens derived from the three selected strains coveredmost of the 397 remaining S aureus field isolates. In other words, itwas expected that a vaccine containing antigens derived from the threeselected strains would induce the production of antibodies that wouldcross react with and protect against most of the 400 isolates.

The group of 400 strains was isolated as follows: Duplicate quarterforemilk samples, obtained from cows that were known to be chronicallyinfected with bovine mastitis, were taken aseptically according to“Laboratory methods for use in mastitis work” (International DairyFederation, Document 132, Brussels, Belgium) and then submitted to thelaboratory.

The bacteriological analysis was performed according to the standardsset forth in the Laboratory and Field Hand Book on Bovine Mastitis,National Mastitis Council, (WI: W. H. Hoard and Sons Co., 1987). 0.01 mlfrom each milk sample was spread over blood-agar plates (Bacto-Agar;Difco Laboratory) containing 5% washed sheep red blood cells. Theminimal detection limit was 5 colony-forming units. The Bacteria wereclassified as S. aureus according to their morphology: 1-3 mm indiameter of bacterial colony; circular, smooth, and raised with abutyrous consistency; type of hemolysis on blood agar cultivated onselective media (Baird Parker and Toluidin Blue DNase); a coagulase test(in rabbit plasma) (Coagulative enzyme); agglutination (specificantibodies against S. aureus) (Remel Santa Fe, Kans., U.S.A.); phagetyping using the international set of typing phages for human strains(Blair and Williams, Bull. Wld. Hlth. Org. 1961, 24, 771-784). Thebiochemical and enzyme characterization was performed by ID-32 API STAPH(Bio Merieux Vitex, Inc., MO, U.S.A.). An antibiogram test was alsoperformed.

In another aspect, the present invention provides a vaccine against theoccurrence of bovine mastitis infection, which vaccine comprises acombination of antigens derived from the S. aureus strains. In anexemplary embodiment, a combination of antigens derived from the S.aureus strains is admixed with an adjuvant, such as an incompleteFreund's adjuvant (IFA) (Difco), for example. In another embodiment, thevaccine is a combination of 0.33 ml of each of the S. aureus strainswith the incomplete Freund's adjuvant in a ratio of 1:1, giving a finalvolume of 2 ml. This vaccine protects challenged cows from udder diseasefollowing intermammary infection with S. aureus.

In accordance with a further aspect, the present invention provides amethod for stimulating a cow's immune system to respond to an antigenderived from the S. aureus strains by administering a vaccine preparedin accordance with the invention. In one embodiment, the vaccine isadministered to heifers about 50 to 60 days before the first parturitionand then boosted (i.e., re-administered) about 30 days thereafter (i.e.,after the first parturition). In another embodiment, the cows arevaccinated 30 days before the second and/or any consecutive parturition.In one embodiment, the cows are vaccinated subcutaneously (sc), underthe tail root and in the area of the supra mammary lymph node.

The various aspects of the invention will now be illustrated withreference to the following non-limiting examples and the figures:

EXAMPLE 1

Separation of the S. aureus Crude Extract

Culture medium: Colombia broth (Difco) was modified by the addition of0.1% d-glucose, 1% yeast extract and 0.5% NaCl. See, Lee, J. C. et al,1987, Infection and Immunity, pp. 2191-2197. The medium was autoclavedat 115° C. for 15 mm., then incubated for 24-28 hr at 37° C., and thenchecked for the sterility of the culture.

Each of the three S. aureus strains (BS449, ZO3984 and VLVL8407) wasgrown for 24 hr at 37° C. in a 5-liter Erlenmeyer containing I liter ofthe medium. At the end of the growth period the broth was checked forthe purity of the culture.

The BS449 and ZO3984 bacterial strains were harvested by centrifugation(3000×g, for 15 min. at 4° C.) and then washed 3 times in phosphatebuffered saline (PBS) at pH 7.2.

The bacteria pellets [3.3 g/L (wet weight)] were each suspended inapproximately 500 ml of PBS and subjected to mechanical agitation withglass beads by cell homogenizer (B. Braun Melsungen AG, Germany) for10-15 minutes. The glass beads and the remaining bacteria were removedby centrifugation and discarded. The remaining solution was filteredthrough 0.80 and 0.45 μm-pore-size membranes. The following enzymes wereadded to the filtrate solution: 250 μg/100 ml of DNase and 750μg/100 mlRNase (Worthington Diagnostics, Freehold, N.J.), which was thenincubated for 1 hr at 37° C. After incubation 2 ml of 0.1 mMphenylmethylsulfonyl fluoride (Sigma) in Acetone and 0.2 ml of 10 mMTosyl (Sigma) were added to a 100 ml solution. Each solution was checkedfor the sterility of the culture. The protein concentrate in thesolution was assayed by Braedford (Bio-Rad, UK) and according to theconcentration (±0.3 mg/ml), the solution of each bacteria was aliquotedand kept at −80° C. The bacteria solutions of strains BS449 and ZO3984were marked (BSs) and (ZOs), respectively.

The culture supernatant of the VLVL8407 strain (marked VLs) wascentrifugated (3000×g for 15 mm. at 4° C.) and then filtered through0.45 μm-pore-size membranes and finally concentrated to approximately1:10 from the original volume. The concentration was performed in acellulose tubular membrane (Nominal MWCO: 3500) (Cellu. Sep. Texas, USA)by Polyethylene glycol 35,000 (Fluka, Switzerland) at 4° C. At the end,the concentrate of the supernatant (VLs) was dialyzed against PBS (pH7.2, 4° C., 48 hr). The VLs was checked for the sterility of theculture. The protein concentrate in the VLs was assayed by Braedford(Bio-Rad, UK) and according to the concentrations (±0.3 mg/ml) wasaliquoted and kept at −80° C.

The antigen analysis of BSs, ZOs, and VLS was performed bypolyacrylamide gel electrophoresis (PAGE) for protein (Lemili, U.K.,1970, “Cleavage of the structural proteins during the assembly of thehead of bacteriophage T4”, Nature, 227:680), and the glycoproteinprofile was derived by an Immuno-blot kit (Bio-Rad).

EXAMPLE 2

Vaccine Preparation and Vaccination

Prior to the administration of the vaccine, antigens derived from theBSs, ZOs, and VLs were thawed, and 0.33 ml of each was mixed 1:1 withincomplete Freund's adjuvant (IFA) (Difco) to give a final volume of 2ml/cow. Each cow was vaccinated subcutaneously (sc), 1 ml under the tailroot and 1 ml in the area of the supra mammary lymph node.

The vaccine was administered to heifers about 50 to 60 days before thefirst parturition and then boosted (i.e., re-administered) about 30 daysthereafter (i.e., after the first parturition). Cows were alsovaccinated 30 days before the second parturition.

EXAMPLE 3

Toxicity and Efficacy of the Vaccines

The toxicity of the vaccine (each batch) was tested in a group of 8 mice(BALB/C). Mice were inoculated intraperitoneally (IP) with 1 ml ofantigens derived from BSs, ZOs and VLs, mixed, as described above. Themice were under surveillance for 1 week. None of these mice showed anysymptoms of toxicity, no mortality or morbidity.

The efficacy of the vaccine was tested by vaccinating the mice sc with0.1 ml of the final vaccine (with IFA). The sera of the mice (prior toand post vaccination) were tested for antibodies against the BS, ZO, andVL antigens by Westernblot (Towbin, H., Staehelin, T. and Gordon, J.1979). Electrophoretic transfer of proteins from polyacrylamide gels tonitrocellulose sheets: for procedures and some applications, see Proc.Natl. Acad. Sci., USA, 76:4350.

Illustrative results of administering the vaccine to mice are summarizedin FIG. 1.

EXAMPLE 4

Two consecutive experiments were conducted, I and II. Each experimentincluded 10 Israeli Holstein cows in mid-lactation, yielding about 25-35Kg/day milk free of bacterial infection or contaminated with minorpathogens and containing low Somatic Cell Counts (SCC) (<300×103/ml). Ineach experiment, the cows were divided into two groups according to theperiod of time from the last parturition, milk yield, SCC, and thestatus of the udder contamination. Before vaccination, blood and milkwere collected from each cow and were tested for specific antibodies byimmunoblot.

The cows in group 1 were vaccinated with the vaccine (BSs, ZOs, VLsmixed 1:1 with IFA, as described above). 1-1.5 ml of the vaccine wasinjected subcutaneously (sc) under the tail root and an additional 1-1.5ml was administered sc in the area of the supra mammary lymph node. Thecows in group 2 were injected similarly with IFA+PBS. The time ofvaccination, boosts, blood collection, bacteriology examination,determination of SCC, and antibody in milk or blood are summarized inFIG. 2 for both experiments.

The cows were boosted once in Exp. I, and twice in Exp. II. The cowswere challenged with 1000 CFU/quarter with S. aureus VLVL8407, each intwo quarters. Milk and blood samples were collected during the postchallenge period in order to examine the bacteriologic status,determined by SCC and antibody levels (FIG. 2).

After the termination of the experiments, sections of the injection areawere submitted to histopathology.

Results

The percentage of the infected quarters and of the cows, two weeks afterchallenge, are summarized in FIG. 3. In Exp. II, one of the vaccinatedcows was excluded before challenge due to drying off (next parturition).

In Exp. I {fraction (8/10)} quarters of the control group and only{fraction (3/9)} quarters in the vaccinated group were shedding S.aureus. In Exp. II, {fraction (11/11)} quarters of the control groupshed S. aureus, and only ⅜ in the vaccinated group did so.

Combining the two experimental groups, {fraction (19/21)} quarters ofthe control group shed S. aureus while only {fraction (6/17)} quartersof the vaccinated cows did so. The difference between vaccinated cowsand control cows, according to the number of quarters shedding S.aureus, was statistically significant (P=0.001). The results aresummarized in FIG. 3.

Two weeks after challenge, the SCC for the quarters that shed S. aureuswere 540×10³ ({fraction (19/21)}) in the control group in comparison to100×10³ ({fraction (6/17)}) in the vaccinated group. This difference ishighly statistically significant (P<0.0001). The results are summarizedin FIG. 4.

The histopathological examination of the tissues around the injectedarea revealed normal structure with no pathological findings.

We claim:
 1. A Staphylococcus aureus strain selected from the groupconsisting of S. aureus BS449, S. aureus ZO3984, and S. aureus VLVL8407.2. A composition comprising a combination of at least two Staphylococcusaureus strains selected from the group consisting of S. aureus BS449, S.aureus ZO3984, and S. aureus VLVL8407.
 3. The Staphylococcus aureusstrain of claim 1, wherein said S. aureus BS449 is beta-hemolytic,coagulase positive and phage type
 81. 4. The Staphylococcus aureusstrain of claim 1, wherein said S. aureus ZO3984 is beta-hemolytic,coagulase positive and phage type 3/A, 3/C, 55 and
 71. 5. TheStaphylococcus aureus strain of claim 1, wherein said S. aureus VLVL8407is non-hemolytic and coagulase positive.
 6. A vaccine against theoccurrence of bovine mastitis, said vaccine comprising a combination ofat least two Staphylococcus aureus strains selected from the groupconsisting of S. aureus BS449, S. aureus ZO3984 and S. aureus VLVL8407.7. The vaccine of claim 6, further comprising an adjuvant.
 8. Thevaccine of claim 7, wherein said adjuvant is an Incomplete FreundAdjuvant.
 9. A method of vaccinating an animal against the occurrence ofbovine mastitis, said method comprising the step of: administering tosaid animal a vaccine comprising a combination of antigenic compositionsextracted from at least two Staphylococcus aureus strains selected fromthe group consisting of S. aureus BS449, S. aureus ZO3984, and S. aureusVLVL8407.
 10. The method of claim 7, wherein vaccine is administered tosaid animal subcutaneously under the tail root and in the area of theanimal's supra lymph node.